Using recently developed DNA mediated gene transfer techniques and molecular cloning methods, this project investigates the identity and multiplicity of transforming genes activated by different carcinogens in various target cells, including epithelial cells. DNA transfection procedures are used to study transformation of different recipient cell lines (NIH/3T3, BALB/3T3, cl A31-1-1 and C3H10Tl/2 cl 8) by high molecular weight DNA extracted from cell lines transformed by several different carcinogens. Preliminary positive results in NIH/3T3 cells were obtained with DNA from 3 out of 4 lines of BALB/3T3 cells transformed by benzo[a]pyrene, and with DNA from hamster cells transformed by 4-nitroquinoline-l-oxide. Restriction enzyme analysis is used to characterize the transforming DNA's. In collaboration with the Cell Biology Section, LVC, NCI, a P-3 laboratory unit was established. In order to study effects of carcinogens on cellular gens, methods are being set up for cloning prospective genes in plasmid and phage vectors. The transforming DNA was isolated from a mouse epidermal cell line (JB-6) transformed by the promoting agent 12-0-tetradecanoylphorbol-13-acetate (TPA) and this DNA was shown to be expressed in the transformed cells since it was DNAse I hypersensitive. Sensitivity to transformation induction by tumor promoters, characteristic of promoter-sensitive subclones of the JB-6 mouse epidermal cell line, was shown to be transferable to promoter-resistant subclones by transfection of DNA from the sensitive cells. The identity of the gene or gene responsible for this property of epithelial cells is being investigated. In addition, a human genome library from sperm DNA was constructed in the Cheron 4A phage vector.